Osteoporosis is a major adverse effect of glucocorticoid (GC) treatment for the amelioration of symptoms of autoimmune and inflammatory diseases. The most important single mechanism contributing to bone loss in GC-treated patients is inhibition of osteoblastic bone formation. We recently identified a commitment stage during osteoblast differentiation in culture (the "cobblestone" stage) in which GC exert their inhibitory effect. During this stage, proliferation persists, but is regulated uniquely compared to control of the cell cycle prior to confluence. At this commitment stage, but not earlier, GC inhibit cell cycle progression and this inhibition is mediated by attenuation of the Ser9 inhibitory phosphorylation of Glycogen Synthase Kinase 3beta(GSKbeta). GSK3beta3 plays pivotal roles in cell proliferation and differentiation. It has been implicated in both the Wnt beta - catenin pathway and the growth factor-activated PI3 kinase/Akt pathway. Numerous studies demonstrated roles for growth factors such as IGF-I and FGF-2 in osteoblast differentiation, but the involvement of GSK3beta has not been sufficiently studied. Several recent reports suggest a role for Wnt signaling in osteoblast differentiation, but again the involvement of GSK3beta has not been tested. Thus, we propose to examine the role of GSK3beta in the osteoblast differentiation-related cell cycle, in the development of the osteoblast phenotype and in GC-mediated inhibition of these processes. In Specific Aim 1, we will examine the role that GSK3beta plays down stream of PI3 Kinase and Akt. In Specific Aim 2, we will examine the role that GSK3beta plays downstream of Wnt. In both Specific Aims, we will measure levels and activities of various components of each pathway in GC-treated versus non-treated osteoblast cultures. In addition, to map the step that is directly affected by GC, we will either augmnent or suppress signaling molecules in each pathway, and examine the effects on GSK3beta, the cell cycle as well as differentiation markers in GC-treated and non-treated cultures. These studies will shed light on novel aspects of osteoblast growth and differentiation, and how these processes are adversely affected by glucocorticoids.